Rap proteins comprise a homologous family of cytoplasmic proteins that regulate bacterial gene expression via remarkably different mechanisms. Secreted signals, called Phr peptides, are imported into the cell where they bind to Rap proteins and repress their activities. The overall goal of our research is to determine how Rap-Phr signaling systems function mechanistically to regulate bacterial signal transduction. One subset of Rap proteins negatively regulates sporulation in B. subtilis by increasing the rate at which Spo0F, a central protein in the sporulation signal transduction pathway, catalyzes the dephosphorylation of a regulatory aspartic acid. Another subset of Rap proteins downregulates the development of genetic competence in B. subtilis by inhibiting ComA, the master transcriptional regulator of early competence gene expression, from binding to target DNA promoters. Additional B. subtilis Rap proteins that are not subjects of immediate study in this proposal regulate the mobility of genetic elements and antagonize the activity of transcription factors other than ComA. It is important from a public health standpoint to determine how Rap proteins regulate bacterial signal transduction because Rap proteins regulate virulence phenotypes in pathogenic organisms. For example, sporulation is repressed in Bacillus anthracis, the causative agent of the disease anthrax, by Rap proteins encoded on its chromosome and virulence plasmid, pX01. This repression is required for B. anthracis to become pathogenic vegetative cells in the infected host. How Rap proteins function mechanistically to regulate the diverse activities of their target proteins is not understood. Interestingly, Phr peptides are generated by an export maturation pathway from small proteins encoded by genes that overlap with the 3 end of the rap genes. Mature Phr pentapeptide molecules are imported into the cell where they bind to Rap proteins and inhibit their negative regulatory effects on gene expression. In Aim 1 we will determine how RapC negatively regulates genetic competence in B. subtilis by inhibiting the binding of ComA to target DNA promoters and also show how the secreted signal, PhrC, promotes genetic competence by inhibiting the interaction of RapC and ComA. In Aim 2 we will reveal how RapA inhibits B. subtilis sporulation by increasing the rate of Spo0F dephosphorylation and also determine how the secreted signal, PhrA, induces sporulation by inhibiting the interaction of RapA and Spo0F. The X-ray crystallographic, biochemical, and bacterial genetic studies proposed here will reveal, for the first time, how Rap proteins regulate the activities of their target proteins and how Phr peptides inhibit Rap protein function. Revealing the molecular mechanisms of Rap-Phr function will enable us to accomplish our long-term goal of designing antibacterial drugs that modulate bacterial signal transduction. PUBLIC HEALTH RELEVANCE Unique cellular proteins modulate the growth, proliferation, and virulence of bacteria, including common human pathogens. Bacterial infections are becoming increasingly difficult to treat, and an escalating threat to public health, as they acquire resistance to existing antibacterial drugs. The long-term goal of our work is to use biochemical, biophysical, and genetic approaches to elucidate the functions of bacterial regulatory proteins, and to design new classes of antibacterial drugs that target these proteins.